Kevin C. Vaughn

Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L.

The seed coats of S. spinosa (prickly sida, Malvaceae) become impermeable to water during seed development on the mother plant. After the seeds have dehydrated during the final maturation stages, piercing of seed coats is necessary to induce imbibition of water and germination. Onset of impermeability occurs during seed coat browning, well in advance of seed dehydration. I. Marbach and A.M. Mayer (1975, Plant Physiol. 56, 93–96) implicated polyphenol oxidase (PO; EC 1.10.3.1) as catechol oxidase in the formation of insoluble polymers during development of coat impermeability in a wild strain of pea (Pisum elatius) seeds. We found, however, that peroxidase (EC 1.11.1.7), not PO, is instrumental in the development of water-impermeable seed coats in prickly sida. We isolated coats and embryos from seeds harvested at several stages of development. Highest peroxidase activity of coat extracts correlated well with the developmental stages of maximum conversion of soluble phenolics to insoluble lignin polymers. Although seed extracts oxidized dihydroxyphenylalanine, this activity was eliminated by catalase, indicating that the oxidation of phenolics in the coat is catalyzed by peroxidase rather than PO. Histochemical localization of peroxidase was strongest in the palisade layer; both the level and time of appearance of activity was proportional to the spectrophotometric assays of seed-coat extracts. The presence of peroxidase and the absence of PO in the seed coat were also confirmed with immunocytochemistry. Our results support the view that peroxidase is involved in the polymerization of soluble phenolics to insoluble lignin polymers during development of prickly sida seed coats, causing the formation of a water-impermeable barrier prior to seed dehydration. As dehydration proceeds, the chalazal area finally becomes impermeable resulting in the hard mature seeds of prickly sida.

Microtubule-Organizing Centers and Nucleating Sites in Land Plants

Microtubule-organizing centers (MTOCs) are morphologically diverse cellular sites involved in the nucleation and organization of microtubules (MTs). These structures are synonymous with the centrosome in mammalian cells. In most land plant cells, however, no such structures are observed and some have argued that plant cells may not have MTOCs. This review summarizes a number of experimental approaches toward the elucidation of those subcellular sites involved in microtubule nucleation and organization. In lower land plants, structurally well-defined MTOCs are present, such as the blepharoplast, multilayered structure, and polar organizer. In higher plants, much of the nucleation and organization of MTs occurs on the nuclear envelope or other endomembranes, such as the plasmalemma and smooth (tubular) endoplasmic reticulum. In some instances, one endomembrane may serve as a site of nucleation whereas others serve as the site of organization. Structural and motor microtubule-associated proteins also appear to be involved in MT nucleation and organization. Immunochemical evidence indicates that at least several of the proteins found in mammalian centrosomes, gamma-tubulin, centrin, pericentrin, and polypeptides recognized by the monoclonal antibodies MPM-2, 6C6, and C9 also recognize putative lower land plant MTOCs, indicating shared mechanisms of nucleation/organization in plants and animals. The most recent data from tubulin incorporation in vivo, mutants with altered MT organization, and molecular studies indicate the potential of these research tools in investigation of MTOCs in plants.

Dodder hyphae invade the host: a structural and immunocytochemical characterization

Summary. Dodder (Cuscuta pentagona) hyphae are unique amongst the parasitic weeds for their ability to apparently grow through the walls of the host plant. Closer examination reveals, however, that the hyphae do not grow through the host but rather induce the host to form a new cell wall (or extend the existing wall) to coat the growing hypha. This chimeric wall composed of walls from two species is even traversed by plasmodesmata that connect the two cytoplasms. Compositionally, the chimeric wall is quite different from the walls of either the host or in other cells of the dodder plant, on the basis of immunocytochemical labeling. The most striking differences were in the pectins, with much stronger labeling present in the chimeric wall than in either the host or other dodder walls. Interestingly, labeling with monoclonal antibodies specific to arabinan side chains of rhamnogalacturonan I pectin fraction was highly enriched in the chimeric wall, but antibodies to galactan side chains revealed no labeling. Arabinogalactan protein antibodies labeled the plasma membrane and vesicles at the tips of the hyphae and the complementary host wall, although the JIM8-reactive epitope, associated with very lipophilic arabinogalactan proteins, was found only in dodder cells and not the host. Callose was found in the plasmodesmata and along the forming hyphal wall but was found at low levels in the host wall. The low level of host wall labeling with anticallose indicates that a typical woundlike response was not induced by the dodder. When dodder infects leaf lamina, which have more abundant intercellular spaces than petioles or shoots, the hyphae grew both intra- and extracellularly. In the latter condition, a host wall did not ensheath the parasite and there was clear degradation of the host middle lamellae by the growing hyphae, allowing the dodder to pass between cells. These data indicate that the chimeric walls formed from the growth of the host cell wall in concert with the developing hyphae are unique in composition and structure and represent an induction of a wall type in the host that is not noted in surrounding walls.

Immunocytochemical characterization of tension wood: Gelatinous fibers contain more than just cellulose.

Gelatinous fibers (G-fibers) are the active component of tension wood. G-fibers are unlike traditional fiber cells in that they possess a thick, nonlignified gelatinous layer (G-layer) internal to the normal secondary cell wall layers. For the past several decades, the G-layer has generally been presumed to be composed nearly entirely of crystalline cellulose, although several reports have appeared that disagreed with this hypothesis. In this report, immunocytochemical techniques were used to investigate the polysaccharide composition of G-fibers in sweetgum (Liquidambar styraciflua; Hamamelidaceae) and hackberry (Celtis occidentalis; Ulmaceae) tension wood. Surprisingly, a number of antibodies that recognize arabinogalactan proteins and RG I-type pectin molecules bound to the G-layer. Because AGPs and pectic mucilages are found in other plant tissues where swelling reactions occur, we propose that these polymers may be the source of the contractile forces that act on the cellulose microfibrils to provide the tension force necessary to bend the tree trunk.

Gelatinous fibers are widespread in coiling tendrils and twining vines

Although the coiling of tendrils and the twining of vines has been investigated since Darwins time, a full understanding of the mechanism(s) of this coiling and twining ability has not yet been obtained. In a previous study (Planta 225: 485-498), gelatinous (G) fibers in tendrils of redvine occurred concomitantly with the ability to coil, strongly indicating their role in the coiling process. In this study, tendrils and twining vines of a number of species were examined using microscopic and immunocytochemical techniques to determine if a similar presence and distribution of these fibers exists in other plant species. Tendrils that coiled in many different directions had a cylinder of cortical G fibers, similar to redvine. However, tendrils that coiled only in a single direction had gelatinous fibers only along the inner surface of the coil. In tendrils with adhesive tips, the gelatinous fibers occurred in the central/core region of the tendril. Coiling occurred later in development in these tendrils, after the adhesive pad had attached. In twining stems, G fibers were not observed during the rapid circumnutation stage, but were found at later stages when the vines position was fixed, generally one or two nodes below the node still circumnutating. The number and extent of fiber development correlated roughly with the amount of torsion required for the vine to ascend a support. In contrast, species that use adventitious roots for climbing or were trailing/scrambling-type vines did not have G fibers. These data strongly support the concept that coiling and twining in vines is caused by the presence of G fibers.

Sequence of effects of acifluorfen on physiological and ultrastructural parameters in cucumber cotyledon discs

Abstract The time course for development of herbicidal effects of acifluorfen on the ultrastructure and physiology of cotyledon discs from 6- to 8-day-old, light-grown cucumber ( Cucumis sativus L.) seedlings was examined. After 20 hr of incubation in darkness in 30 μ M acifluorfen no herbicidal damage was detected, but within 1.5 hr after subsequent exposure to 420 μEin m −2 sec −1 PAR, chloroplast envelope, tonoplast, and plasma membrane disruption was observed. Choloroplast thylakoid disruption occurred after envelope disruption. CO 2 -Dependent O 2 evolution was reduced ca. 30% within 1 hr of the onset of light exposure, whereas dark O 2 uptake was not affected during a 5-hr time course. Reduction of in vivo variable chlorophyll fluorescence and cytochrome f oxidation/reduction was observable within 1 hr and was reduced by ca. 90% within 5 hr. Neither carotenoid nor chlorophyll content declined significantly during 5 hr of light, whereas acifluorfen caused increases in ethane, ethylene, and malondialdehyde within 2 hr. Increased leakage of electrolytes was detectable within 1 hr. These data indicate that loss of membrane integrity, perhaps beginning with the plastid envelope, precedes most of the other symptoms of acifluorfen-caused cellular damage.

The herbicide dichlobenil disrupts cell plate formation: immunogold characterization

SummaryWe have utilized light and transmission electron microscopy and immunocytochemistry to examine onion roots treated with the herbicide dichlobenil (2,6-dichlorobenzonitrile; DCB), a purported disrupter of cellulose biosynthesis. The most salient effect of DCB is observed on cell plate formation, the process that gives rise to new cell walls. In the presence of DCB, cell plates develop normally up to the tubular network stage. They are the result of fusion of Golgi-derived vesicles and the accumulation of callose and the first strands of cellulose. The DCB-treated cell plates retain the reticulate and malleable nature of the tubular network/early fenestrated plate stage of cell plate formation, but fail to display signs of the stiffening and straightening associated with an accumulation of cellulose. Instead, the malleable cell plates in the DCB-treated cells retain a wavy architecture, accumulate pockets of electron opaque material, and produce plasmodesmata in abnormal orientations. Immunocytochemical investigations of the abnormal cell plates formed after DCB treatment show 20-fold increase in the level of callose labelling found in the control cell plates. Xyloglucans and rhamnogalacturonans can be detected in the partially-formed cell plates, with the labelling density of xyloglucan 4–5 times greater than in the control cell plates and that of the rhamnogalacturonans being similar to the controls. These data support the hypothesis that DCB inhibits cellulose biosynthesis as a primary mechanism of action, and that in the absence of cellulose synthesis the cell plates fail to mature and to give rise to new cross walls.

A limited survey of the phylogenetic distribution of polyphenol oxidase

Abstract A variety of terrestrial and aquatic plant species were tested for the presence of polyphenol oxidase (PPO), using three different assay protocols. All aquatic green algae ( Chlorella , Stigeoclonium , Microspora , Ulva and Spirogyra ) tested have no detectable PPO except for a Trebouxioid alga (lichen symbiont), and Coleochaete . Of the Charalean algae Chara and Nitella , only the latter had measurable activity. Some hepatics ( Conocephalum and Marchantia) possess the enzyme, but two species of Riccia tested do not. The anthocerotes Phaeoceros , Anthoceros and Notothylas have low, but easily detectable PPO. The mosses, Dicranum , Sphagnum and Thuidium , have no detectable activity. All of the ferns and fern allies tested have PPO activity. Among seed plants, PPO is undetectable in Ginkgo or any of the conifers tested. All but two species of the angiosperms surveyed possessed the enzyme. The M r as determined by semi-native electrophoretic mobility, ranged widely from 30 000 to 200 000, depending upon species. All species with PPO activity measured by spectrophotometric or gel assay had cytochemically-detected activity localized to the thylakoid membranes, with cytochemical product accumulating in the lumen of the thylakoid.

Mitotic disrupter herbicides act by a single mechanism but vary in efficacy

SummaryAlthough there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different “mitotic disrupter herbicides”, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.

A cortical band of gelatinous fibers causes the coiling of redvine tendrils: a model based upon cytochemical and immunocytochemical studies.

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S1 and S2) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.